我们感谢人类遗传学研究所内的工作人员对图书馆的准备和测序。我们感谢 Einat Snir 在为这里制作的 Y2H 质粒库编写基因组文库片段方面的专长。这项工作得到国家卫生研究院的支持: NIH R21 EB021870-01A1 和 NSF 研究项目赠款: 1517110。

<ol>
	<li>Fields, S., Song, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+genetic+system+to+detect+protein-protein+interactions.">A novel genetic system to detect protein-protein interactions.</a> Nature. 340 (6230), 245-246 (1989).</li><li>Bruckner, A., Polge, C., Lentze, N., Auerbach, D., Schlattner, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Yeast+two-hybrid,+a+powerful+tool+for+systems+biology.">Yeast two-hybrid, a powerful tool for systems biology.</a> International Journal of Molecular Sciences. 10 (6), 2763-2788 (2009).</li><li>Vidal, M., Fields, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+yeast+two-hybrid+assay:+still+finding+connections+after+25+years.">The yeast two-hybrid assay: still finding connections after 25 years.</a> Nature Methods. 11 (12), 1203-1206 (2014).</li><li>Pashkova, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DEEPN+as+an+Approach+for+Batch+Processing+of+Yeast+2-Hybrid+Interactions.">DEEPN as an Approach for Batch Processing of Yeast 2-Hybrid Interactions.</a> Cell Reports. 17 (1), 303-315 (2016).</li><li>Rajagopala, S. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mapping+the+Protein-Protein+Interactome+Networks+Using+Yeast+Two-Hybrid+Screens.">Mapping the Protein-Protein Interactome Networks Using Yeast Two-Hybrid Screens.</a> Advances in Experimental Medicine and Biology. 883, 187-214 (2015).</li><li>Weimann, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Y2H-seq+approach+defines+the+human+protein+methyltransferase+interactome.">A Y2H-seq approach defines the human protein methyltransferase interactome.</a> Nature Methods. 10 (4), 339-342 (2013).</li><li>Yachie, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pooled-matrix+protein+interaction+screens+using+Barcode+Fusion+Genetics.">Pooled-matrix protein interaction screens using Barcode Fusion Genetics.</a> Molecular Systems Biology. 12 (4), 863 (2016).</li><li>Trigg, S. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CrY2H-seq:+a+massively+multiplexed+assay+for+deep-coverage+interactome+mapping.">CrY2H-seq: a massively multiplexed assay for deep-coverage interactome mapping.</a> Nature Methods. , (2017).</li><li>Estojak, J., Brent, R., Golemis, E. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Correlation+of+two-hybrid+affinity+data+with+in+vitro+measurements.">Correlation of two-hybrid affinity data with in vitro measurements.</a> Molecular and Cellular Biology. 15 (10), 5820-5829 (1995).</li><li>Rajagopala, S. V., Hughes, K. T., Uetz, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Benchmarking+yeast+two-hybrid+systems+using+the+interactions+of+bacterial+motility+proteins.">Benchmarking yeast two-hybrid systems using the interactions of bacterial motility proteins.</a> Proteomics. 9 (23), 5296-5302 (2009).</li><li>James, P., Halladay, J., Craig, E. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genomic+libraries+and+a+host+strain+designed+for+highly+efficient+two-hybrid+selection+in+yeast.">Genomic libraries and a host strain designed for highly efficient two-hybrid selection in yeast.</a> Genetics. 144 (4), 1425-1436 (1996).</li><li>Barbas, C. F., Burton, D. R., Scott, J. K., Silverman, G. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitation+of+DNA+and+RNA.">Quantitation of DNA and RNA.</a> CSH Protoc. 2007, pdb ip47 (2007).</li><li>Sambrook, J., Russell, D. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=SDS-Polyacrylamide+Gel+Electrophoresis+of+Proteins.">SDS-Polyacrylamide Gel Electrophoresis of Proteins.</a> Cold Spring Harbor Protocols. 2006 (4), (2006).</li><li>Towbin, H., Staehelin, T., Gordon, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrophoretic+transfer+of+proteins+from+polyacrylamide+gels+to+nitrocellulose+sheets:+procedure+and+some+applications.">Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications.</a> Proceedings of the National Academy of Sciences USA. 76 (9), 4350-4354 (1979).</li><li>Alegria-Schaffer, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Western+blotting+using+chemiluminescent+substrates.">Western blotting using chemiluminescent substrates.</a> Methods in Enzymology. 541, 251-259 (2014).</li><li>Lee, P. Y., Costumbrado, J., Hsu, C. Y., Kim, Y. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Agarose+gel+electrophoresis+for+the+separation+of+DNA+fragments.">Agarose gel electrophoresis for the separation of DNA fragments.</a> Journal of Visualized Experiments. (62), (2012).</li><li>Harper, J. W., Adami, G. R., Wei, N., Keyomarsi, K., Elledge, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+p21+Cdk-interacting+protein+Cip1+is+a+potent+inhibitor+of+G1+cyclin-dependent+kinases.">The p21 Cdk-interacting protein Cip1 is a potent inhibitor of G1 cyclin-dependent kinases.</a> Cell. 75 (4), 805-816 (1993).</li></ol>

作者没有什么可透露的

在这里, 我们提供了如何使用优化方法在批处理中执行 Y2H 化验的指南。在这个过程中有几个关键步骤, 以帮助确保将被放置在选择下的酵母的人口是起始库的代表, 并且足够的开始酵母人口用于接受选择限制变异.重要的是, 这些基准相对容易实现, 同时适应传统的 Y2H 检测方法和材料, 从而使大多数实验室都能使用标准分子生物学的方法。在使用不同的诱饵质粒时, DEEPN 允许从相同的猎物库种群中进...

对细胞生物学过程的完全理解依赖于寻找其分子机制基础的蛋白质相互作用网络。一种识别蛋白质相互作用的方法是酵母 2-杂交 (Y2H) 法, 它通过组装一个功能的嵌合体转录因子, 一旦两个感兴趣的蛋白质领域绑定到另一个1。一个典型的 Y2H 屏幕是通过创建一个酵母种群, 它既可以将相互作用的蛋白质的质粒库编码成转录激活剂 (e. g, "猎物" 融合蛋白) 和由蛋白质组成的给定的 "诱饵" 质粒。的兴趣融合到 dna 绑定域 (例如, 绑定到 Gal4-upstream 激活序列的 Gal4 DNA 绑定域)。Y2H 方法的一个主要优点是, 在为常规分子生物学工作配备的典型实验室中, 进行2是相对容易和廉价的。但是, 当传统上执行时, 用户会对在选择时出现的单个菌落进行正面 Y2H 交互的采样。这严重限制了可以调查的图书馆 "猎物" 克隆的数量。当一个特定的相互作用的猎物的丰度相对于其他捕食者非常高时, 这一问题就会复杂化, 从而减少了从低丰度猎物质粒中检测相互作用的几率。
在蛋白质组的综合覆盖范围内使用 Y2H 原理的一个解决方案是使用矩阵格式化的方法, 其中包含已知单个猎物质粒的阵列可以进行数字审问。但是, 这种方法需要的基础结构对于那些有意定义少量蛋白质或域3的 interactome 的个体调查人员来说是不容易接近或成本效益高的。此外, 非常复杂的猎物库, 可能编码多个相互作用的蛋白质片段, 将扩大这种矩阵数组的大小不切实际的大小。另一种方法是在批次中执行复杂库的检测, 并使用大规模并行高通量排序4评估交互克隆的存在。这可以用来检测在多个殖民地出现的猎物质粒的存在, 使用典型的 Y2H 格式化的方法, 其中酵母细胞外壳的相互作用对融合蛋白被允许生长在一个板块5,6。这一总体思路可以增强, 以增加对多个诱饵和猎物组件的查询, 同时7,8。
尽管如此, 许多调查还是需要更简单、更专注于少量蛋白质 "诱饵" 的努力, 并且可以通过对单个复杂的猎物库进行详尽而半定量的查询来获得更多的好处。我们已经开发并验证了一种使用 Y2H 原则在批处理格式4中执行广泛的蛋白质交互研究的方法。这使用特定的猎物质粒的膨胀率作为 Y2H 交互作用的相对强度的代理9。在正常生长或选择性生长条件下, 在种群内的所有质粒的深度测序生成完整的克隆, 产生强弱的 Y2H 相互作用。interactors 可以在多个诱饵质粒中得到和直接比较。由此产生的工作流称为 DEEPN (对蛋白质网络进行动态富集评估), 因此可以用来识别同一个猎物库中的差异 interactomes, 以确定蛋白质, 从而使一种蛋白质与另一种蛋白进行比较。
在这里, 我们演示了 DEEPN, 并介绍了有助于其使用的实验室方法的改进, 其中概述了图 1。重大改进包括:
捕食酵母种群的生成.DEEPN 的关键要求之一是产生不同饵料质粒的酵母种群, 它们的粒细胞分布相同。捕食质粒库的等效基线种群对不同饵料的 interactomes 进行精确比较是必不可少的。这是最好的实现, 当一个图书馆的质粒已经安置在单倍体酵母种群和移动一个给定的诱饵质粒进入该人群是通过交配产生一个倍。在这里, 我们提供了一个明确的指南, 如何使这些人口使用的商业图书馆在单倍体酵母。虽然我们发现了产生大量 diploids 的方法, 但这些含有酵母菌的商业文库的整体交配效率却很低。因此, 我们构造了一种新的应变, 可以使猎物库产生更 diploids 的每一个交配反应。
新的诱饵质粒集。许多目前表达 "诱饵" 融合蛋白的质粒是由感兴趣的蛋白质和 DNA 结合的领域组成的, 2µ, 允许它们放大其拷贝数。这个拷贝数在人口中可能是相当可变的并且导致变异在 Y2H 转录反应。这反过来可能会扭曲的能力, 以衡量一个特定的蛋白质相互作用的强度, 根据细胞的生长反应的选择。这可以通过使用低拷贝质粒来部分解决, 其中一些以前曾被描述为商业上可用的 pDEST3210。我们构建了一种新的诱饵质粒 (pTEF GBD), 在TRP1着丝粒基低拷贝质粒中产生 Gal4-DNA-binding 域融合蛋白, 携带 Kanr 抵抗基因, 同时也允许克隆上游和Gal4 DNA 结合领域的下游。
新的高密度 Y2H 片段库.我们构建了一个新的质粒来 Y2H 猎物库, 并利用它建立一个高度复杂的 Y2H 图书馆由随机剪切片段的基因组 DNA 从酿酒酵母。序列分析表明, 该库有100万多个不同的元素, 远比以前描述的酵母基因组 Y2H 质粒库11复杂得多。通过这个新的库, 我们能够证明 DEEPN 工作流足够健壮, 能够以可靠和可重现的方式容纳许多不同质粒的复杂库。

<table>
	<tbody>
		<tr>
			<td>Illumina HiSeq 4000</td>
			<td>Illumina</td>
			<td></td>
			<td>deep sequencing platform</td>
		</tr>
		<tr>
			<td>Monoclonal anti-HA antibodies</td>
			<td>Biolegend</td>
			<td>901514</td>
			<td>Primary Antibody to detect expression of HA in pGal4AD constructs</td>
		</tr>
		<tr>
			<td>Polyclonal anti-myc antibodies</td>
			<td>QED Biosciences Inc</td>
			<td>18826</td>
			<td>Primary Antibody to detect expression of MYC in pTEF-GBD constructs</td>
		</tr>
		<tr>
			<td>NarI</td>
			<td>New England BioLabs</td>
			<td>R0191S</td>
			<td></td>
		</tr>
		<tr>
			<td>EcoRI-HF</td>
			<td>New England BioLabs</td>
			<td>R3101S</td>
			<td></td>
		</tr>
		<tr>
			<td>BamHI-HF</td>
			<td>New England BioLabs</td>
			<td>R3236S</td>
			<td></td>
		</tr>
		<tr>
			<td>XhoI</td>
			<td>New England BioLabs</td>
			<td>R0146S</td>
			<td></td>
		</tr>
		<tr>
			<td>Polyethylene Glycol 3350, powder</td>
			<td>J.T. Baker</td>
			<td>U2211-08</td>
			<td></td>
		</tr>
		<tr>
			<td>Salmon Sperm DNA</td>
			<td>Trevigen, Inc sold by Fisher Scientific</td>
			<td>50-948-286</td>
			<td>carrier DNA for yeast transformation section 3.2.1.</td>
		</tr>
		<tr>
			<td>Kanamycin Monosulfate</td>
			<td>Research Products International</td>
			<td>K22000</td>
			<td></td>
		</tr>
		<tr>
			<td>LE Agarose</td>
			<td>GeneMate</td>
			<td>E-3120-500</td>
			<td>used for making DNA agarose gels</td>
		</tr>
		<tr>
			<td>Sodium Chloride</td>
			<td>Research Products International</td>
			<td>S23025</td>
			<td></td>
		</tr>
		<tr>
			<td>Tryptone</td>
			<td>Research Products International</td>
			<td>T60060</td>
			<td></td>
		</tr>
		<tr>
			<td>D-Sorbitol</td>
			<td>Research Products International</td>
			<td>S23080</td>
			<td></td>
		</tr>
		<tr>
			<td>Lithium Acetate Dihydrate</td>
			<td>MP Biomedicals</td>
			<td>155256</td>
			<td></td>
		</tr>
		<tr>
			<td>Calcium Chloride</td>
			<td>ThermoFisher</td>
			<td>C79</td>
			<td></td>
		</tr>
		<tr>
			<td>EDTA Sodium Salt</td>
			<td>Research Products International</td>
			<td>E57020</td>
			<td></td>
		</tr>
		<tr>
			<td>Yeast Extract Powder</td>
			<td>Research Products International</td>
			<td>Y20020</td>
			<td></td>
		</tr>
		<tr>
			<td>Yeast Nitrogen Base (ammonium sulfate) w/o amino acids</td>
			<td>Research Products International</td>
			<td>Y20040</td>
			<td></td>
		</tr>
		<tr>
			<td>CSM-Trp-Leu+40ADE</td>
			<td>Formedium</td>
			<td>DCS0789</td>
			<td></td>
		</tr>
		<tr>
			<td>CSM-Trp-Leu-His+40ADE</td>
			<td>Formedium</td>
			<td>DCS1169</td>
			<td></td>
		</tr>
		<tr>
			<td>CSM-Leu-Met</td>
			<td>Formedium</td>
			<td>DCS0549</td>
			<td></td>
		</tr>
		<tr>
			<td>CSM-Trp-Met</td>
			<td>Bio 101, Inc</td>
			<td>4520-922</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Methionine</td>
			<td>Formedium</td>
			<td>DOC0168</td>
			<td></td>
		</tr>
		<tr>
			<td>Adenine</td>
			<td>Research Products International</td>
			<td>A11500</td>
			<td></td>
		</tr>
		<tr>
			<td>D-(+)-Glucose</td>
			<td>Research Products International</td>
			<td>G32045</td>
			<td></td>
		</tr>
		<tr>
			<td>Bacto Agar</td>
			<td>BD</td>
			<td>214010</td>
			<td>used for making media plates in section 1</td>
		</tr>
		<tr>
			<td>Peptone</td>
			<td>Research Products International</td>
			<td>P20240</td>
			<td></td>
		</tr>
		<tr>
			<td>3-amino-1,2,4 Triazole</td>
			<td>Sigma</td>
			<td>A8056</td>
			<td></td>
		</tr>
		<tr>
			<td>2-Mercaptoehanol (BME)</td>
			<td>Sigma-Aldrich</td>
			<td>M6250</td>
			<td></td>
		</tr>
		<tr>
			<td>Zymolyase 100T</td>
			<td>USBiological</td>
			<td>Z1004</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium phosphate dibasic</td>
			<td>Sigma</td>
			<td>P8281</td>
			<td></td>
		</tr>
		<tr>
			<td>Phenol:Chloroform:IAA</td>
			<td>Ambion</td>
			<td>AM9732</td>
			<td></td>
		</tr>
		<tr>
			<td>Ammonium Acetate</td>
			<td>Sigma-Aldrich</td>
			<td>238074</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol</td>
			<td>Decon Laboratories, INC</td>
			<td>2716</td>
			<td></td>
		</tr>
		<tr>
			<td>RNAse A</td>
			<td>ThermoFisher</td>
			<td>EN0531</td>
			<td></td>
		</tr>
		<tr>
			<td>Urea</td>
			<td>Research Products International</td>
			<td>U20200</td>
			<td></td>
		</tr>
		<tr>
			<td>SDS</td>
			<td>Research Products International</td>
			<td>L22010</td>
			<td></td>
		</tr>
		<tr>
			<td>glycerol</td>
			<td>Sigma Aldrich</td>
			<td>G5516</td>
			<td></td>
		</tr>
		<tr>
			<td>Tris-HCl</td>
			<td>Gibco</td>
			<td>15506-017</td>
			<td></td>
		</tr>
		<tr>
			<td>bromophenol blue</td>
			<td>Amresco</td>
			<td>449</td>
			<td></td>
		</tr>
		<tr>
			<td>Gibson Assembly Cloning Kit</td>
			<td>New England Biolabs</td>
			<td>E5510S</td>
			<td>Rapid assembly method for cloning of plasmids in section 2</td>
		</tr>
		<tr>
			<td>NEBNext High-Fidelity 2x PCR Master Mix</td>
			<td>New England Biolabs</td>
			<td>M0541S</td>
			<td>Used for amplification of products for Gibson Assembly in Section 2.3 as well assample preparation for DEEPN deep sequencing in section 6.2.1</td>
		</tr>
		<tr>
			<td>Ethidium Bromide</td>
			<td>Amresco</td>
			<td>0492-5G</td>
			<td></td>
		</tr>
		<tr>
			<td>QIAquick PCR purification kit</td>
			<td>Qiagen</td>
			<td>28104</td>
			<td>Used for purification of pcr products in section 6.2.3</td>
		</tr>
		<tr>
			<td>Qiaquick DNA Gel Extraction Kit</td>
			<td>Qiagen</td>
			<td>28704</td>
			<td>Used for purification of digested pTEF-GBD in section 2.1</td>
		</tr>
		<tr>
			<td>KAPA Hyper Prep kit</td>
			<td>KAPA Biosystems</td>
			<td>KK8500</td>
			<td>preparation kit for deep sequencing</td>
		</tr>
		<tr>
			<td>Codon optimization</td>
			<td>http://www.jcat.de</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Codon optimization</td>
			<td>https://www.idtdna.com/CodonOpt</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>gBlocks</td>
			<td>Integrated DNA Technologies</td>
			<td></td>
			<td>DNA fragments used for cloning in Section 2.2</td>
		</tr>
		<tr>
			<td>Strings</td>
			<td>Thermofisher</td>
			<td></td>
			<td>DNA fragments used for cloning in Section 2.2</td>
		</tr>
		<tr>
			<td>GenCatch Plasmid DNA mini-prep Kit</td>
			<td>EPOCH Life Sciences</td>
			<td></td>
			<td>Used to prepare quantities of DNA in Section 2.3</td>
		</tr>
		<tr>
			<td>Covaris E220</td>
			<td>Covaris</td>
			<td></td>
			<td>high performance ultra-sonicator in section 7</td>
		</tr>
		<tr>
			<td>oligo nucelotide 5&#8217;- CGGTCTT 
			CAATTTCTCAAGTTTCAG -3&#8217;</td>
			<td>Integrated DNA Technologies or Thermofisher</td>
			<td></td>
			<td>used for pcr amplification and sequencing 5&#39; insert pTEF-GBD during plasmid construction</td>
		</tr>
		<tr>
			<td>oligo nucelotide 5&#8217;-GAGTAACG 
			ACATTCCCAGTTGTTC-3&#8217;</td>
			<td>Integrated DNA Technologies or Thermofisher</td>
			<td></td>
			<td>used for pcr amplification and sequencing 5&#39; insert pTEF-GBD during plasmid construction</td>
		</tr>
		<tr>
			<td>oligo nucelotide 5&#8217;-CACCGTAT 
			TTCTGCCACCTCTTCC-3&#8217;</td>
			<td>Integrated DNA Technologies or Thermofisher</td>
			<td></td>
			<td>used for pcr amplification and sequencing 3&#39; insert pTEF-GBD during plasmid construction</td>
		</tr>
		<tr>
			<td>oligo nucelotide 5&#8217;-GCAACCGC 
			ACTATTTGGAGCGCTG-3&#8217;</td>
			<td>Integrated DNA Technologies or Thermofisher</td>
			<td></td>
			<td>used for pcr amplification and sequencing 3&#39; insert pTEF-GBD during plasmid construction</td>
		</tr>
		<tr>
			<td>oligonucleotide 5&#8217;-GTTCCGATG 
			CCTCTGCGAGTG-3&#8217;</td>
			<td>Integrated DNA Technologies or Thermofisher</td>
			<td></td>
			<td>5&#39; Pimer used for insert amplification of pGAL4AD</td>
		</tr>
		<tr>
			<td>oligonucelotide 5&#8217;-GCACATGCT 
			AGCGTCAAATACC-3&#8217;</td>
			<td>Integrated DNA Technologies or Thermofisher</td>
			<td></td>
			<td>3&#39; Pimer used for insert amplification of pGAL4AD</td>
		</tr>
		<tr>
			<td>oligonucelotide 5&#8217;-ACCCAAGCA 
			GTGGTATCAACG-3&#8217;</td>
			<td>Integrated DNA Technologies or Thermofisher</td>
			<td></td>
			<td>5&#39; Pimer used for insert amplification of pGADT7</td>
		</tr>
		<tr>
			<td>oligonucelotide 5&#8217;- TATTTAGA 
			AGTGTCAACAACGTA -3&#8217;</td>
			<td>Integrated DNA Technologies or Thermofisher</td>
			<td></td>
			<td>3&#39; Pimer used for insert amplification of pGADT7</td>
		</tr>
		<tr>
			<td>PJ69-4A MatA yeast strain</td>
			<td>http://depts.washington.edu/yeastrc/ James P, Halladay J, Craig EA: Genomic Libraries and a host strain designed for highly efficient two-hybrid selection in yeast. GENETICS 1996 144:1425-1436</td>
			<td></td>
			<td>MATA leu2-3,112 ura3-52 trp1-901 his3-200 gal4D, gal80D, GAL-ADE2 lys2::GAL1-HIS3 met2::GAL7</td>
		</tr>
		<tr>
			<td>pTEF-GBD</td>
			<td>Dr. Robert Piper Lab</td>
			<td></td>
			<td>Gal4-DNA binding doimain expression plasmid</td>
		</tr>
		<tr>
			<td>PLY5725 MATalpha yeast strain</td>
			<td>Dr. Robert Piper Lab</td>
			<td></td>
			<td>MATalpha his3&#8710;1 leu2&#8710;0 lys2&#8710;0 ura3&#8710;0 gal4&#8710; trp1&#8710; Gal80&#8710;</td>
		</tr>
		<tr>
			<td>pGal4AD (pPL6343)</td>
			<td>Dr. Robert Piper Lab</td>
			<td></td>
			<td>Gal4-activation domain expression plasmid</td>
		</tr>
		<tr>
			<td>100 mm petri dishes</td>
			<td>Kord-Vallmark sold by VWR</td>
			<td>2900</td>
			<td></td>
		</tr>
		<tr>
			<td>125 mL PYREX Erlenmeyer flask</td>
			<td>Fisher Scientific</td>
			<td>S63270</td>
			<td></td>
		</tr>
		<tr>
			<td>250 mL PYREX Erlenmeyer flask</td>
			<td>Fisher Scientific</td>
			<td>S63271</td>
			<td></td>
		</tr>
		<tr>
			<td>1,000 mL PYREX Erlenmeyer flask</td>
			<td>Fisher Scientific</td>
			<td>S63274</td>
			<td></td>
		</tr>
		<tr>
			<td>2,000 mL PYREX Erlenmeyer flask</td>
			<td>Fisher Scientific</td>
			<td>S63275</td>
			<td></td>
		</tr>
		<tr>
			<td>20 X 150 mm Disposable Culture Tube</td>
			<td>Thermofisher</td>
			<td>14-961-33</td>
			<td></td>
		</tr>
		<tr>
			<td>pipet-aid</td>
			<td>Drummond</td>
			<td>4-000-100</td>
			<td></td>
		</tr>
		<tr>
			<td>5 mL Serological Pipette</td>
			<td>Denville</td>
			<td>P7127</td>
			<td></td>
		</tr>
		<tr>
			<td>10 mL Serological Pipette</td>
			<td>Denville</td>
			<td>P7128</td>
			<td></td>
		</tr>
		<tr>
			<td>25 mL Serological Pipette</td>
			<td>Denville</td>
			<td>P7129</td>
			<td></td>
		</tr>
		<tr>
			<td>1,000 mL PYREX Griffin Beaker</td>
			<td>Fisher Scientific</td>
			<td>02-540P</td>
			<td></td>
		</tr>
		<tr>
			<td>1,000 mL PYREX Reuasable Media Storage Bottle</td>
			<td>Fisher Scientific</td>
			<td>06-414-1D</td>
			<td></td>
		</tr>
		<tr>
			<td>1,000 mL graduated cylinder</td>
			<td>Fisher Scientific</td>
			<td>08-572-6G</td>
			<td></td>
		</tr>
		<tr>
			<td>SpectraMax 190</td>
			<td>Molecular Devices</td>
			<td></td>
			<td>used to measure the Optical Density of cells</td>
		</tr>
		<tr>
			<td>NanoDrop 2000</td>
			<td>Thermo Scientific</td>
			<td>ND-2000</td>
			<td>Spectrophotometer used to quantify amount of DNA</td>
		</tr>
		<tr>
			<td>Electronic UV transilluminator</td>
			<td>Ultra Lum</td>
			<td>MEB 20</td>
			<td>used to visualize DNA in an Ethidium Bromide agarose gel</td>
		</tr>
		<tr>
			<td>P1000 Gilson PIPETMAN</td>
			<td>Fisher Scientific</td>
			<td>F123602G</td>
			<td></td>
		</tr>
		<tr>
			<td>P200 Gilson PIPETMAN</td>
			<td>Fisher Scientific</td>
			<td>F123601G</td>
			<td></td>
		</tr>
		<tr>
			<td>P20 Gilson PIPETMAN</td>
			<td>Fisher Scientific</td>
			<td>F123600G</td>
			<td></td>
		</tr>
		<tr>
			<td>P10 Gilson PIPETMAN</td>
			<td>Fisher Scientific</td>
			<td>F144802G</td>
			<td></td>
		</tr>
		<tr>
			<td>1250 &#181;L Low Retention Pipette Tips</td>
			<td>GeneMate</td>
			<td>P-1236-1250</td>
			<td></td>
		</tr>
		<tr>
			<td>200 &#181;LLow Retention Pipette Tips</td>
			<td>VWR</td>
			<td>10017-044</td>
			<td></td>
		</tr>
		<tr>
			<td>10 &#181;L XL Low Retention Pipette Tips</td>
			<td>VWR</td>
			<td>10017-042</td>
			<td></td>
		</tr>
		<tr>
			<td>50 mL conical tube</td>
			<td>VWR</td>
			<td>490001-627</td>
			<td></td>
		</tr>
		<tr>
			<td>15 mL conical tube</td>
			<td>VWR</td>
			<td>490001-621</td>
			<td></td>
		</tr>
		<tr>
			<td>cell scraper</td>
			<td>Denville Scientific</td>
			<td>TC9310</td>
			<td></td>
		</tr>
		<tr>
			<td>1.5 mL Microcentrifuge tubes</td>
			<td>USA Scientific</td>
			<td>1615-5500</td>
			<td></td>
		</tr>
		<tr>
			<td>HCl</td>
			<td>Fluka Analytical</td>
			<td>318949-1L</td>
			<td></td>
		</tr>
		<tr>
			<td>NaOH</td>
			<td>J.T. Baker</td>
			<td>5674-02</td>
			<td></td>
		</tr>
		<tr>
			<td>Wooden applicators</td>
			<td>Solon Care</td>
			<td>55900</td>
			<td></td>
		</tr>
		<tr>
			<td>Eppendorf microcentrifuge 5424</td>
			<td>Fisher Scientific</td>
			<td>05-400-005</td>
			<td>microcentrifuge</td>
		</tr>
		<tr>
			<td>Sorvall ST16R</td>
			<td>Thermo Fisher Scientific</td>
			<td>75004381</td>
			<td>benchtop centrifuge</td>
		</tr>
		<tr>
			<td>Amersham ECL Rabbit IgG, HRP-linked whole Ab (from donkey)</td>
			<td>GE Healthcare</td>
			<td>NA934-1ML</td>
			<td>Secondary Antibody</td>
		</tr>
		<tr>
			<td>Amersham ECL Mouse IgG, HRP-linked whole Ab (from sheep)</td>
			<td>GE Healthcare</td>
			<td>NA931-1ML</td>
			<td>Secondary Antibody</td>
		</tr>
		<tr>
			<td>SuperSignal West Pico Chemiluminescent Substrate</td>
			<td>Thermo Fisher Scientific</td>
			<td>34080</td>
			<td>ECL detection solution</td>
		</tr>
		<tr>
			<td>Isotemp Incubator</td>
			<td>Thermo Fisher Scientific</td>
			<td></td>
			<td>Incubator</td>
		</tr>
		<tr>
			<td>Mutitron 2</td>
			<td>INFORS HT</td>
			<td></td>
			<td>Shaking incubator</td>
		</tr>
		<tr>
			<td>Isotemp Digital-Control Water Bath Model 205</td>
			<td>Fisher Scientific</td>
			<td></td>
			<td>water bath</td>
		</tr>
		<tr>
			<td>Y2H mouse cDNA library in Y187 (pan tissue)</td>
			<td>Clontech</td>
			<td>630482</td>
			<td>commercially available cDNA Library</td>
		</tr>
		<tr>
			<td>Y2H mouse cDNA library in Y187 (mouse brain)</td>
			<td>Clontech</td>
			<td>630488</td>
			<td>commercially available cDNA Library</td>
		</tr>
		<tr>
			<td>pGADT7 AD Vector</td>
			<td>Clontech</td>
			<td>630442</td>
			<td>commercially available AD Vector housing many cDNA libraries</td>
		</tr>
		<tr>
			<td>pGBKT7 DNA-BD Vector</td>
			<td>Clontech</td>
			<td>630443</td>
			<td>commercially available DNA-BD Vector</td>
		</tr>
		<tr>
			<td>Biolase DNA Polymerase</td>
			<td>Bioline</td>
			<td>BIO-21042</td>
			<td>DNA polymerase used for section 2.4</td>
		</tr>
		<tr>
			<td>GeneMate GCL-60 Thermal Cycler</td>
			<td>BioExpress</td>
			<td>P-6050-60</td>
			<td>pcr machine</td>
		</tr>
		<tr>
			<td>TempAssure 0.5 mL PCR tubes</td>
			<td>USA Scientific</td>
			<td>1405-8100</td>
			<td></td>
		</tr>
	</tbody>
</table>

a yeast 2-hybrid screen in batch to compare protein interactions

用酵母 2-杂交法筛选蛋白质-蛋白质相互作用长期以来一直是一个有效的工具, 但它的使用在很大程度上仅限于发现高亲和性的 interactors 在互动的候选库中高度丰富。以传统的形式, 酵母 2-杂交试验可以产生太多的菌落分析时进行低严格的, 在那里可能发现低亲和 interactors。此外, 如果没有对同一图书馆进行全面和彻底的讯问, 对不同的诱饵质粒, 就无法进行比较分析。虽然这些问题中的一些可以使用阵列的牺牲品库来解决, 但操作此类屏幕所需的成本和基础设施却是令人望而却步的。作为替代, 我们已经适应了酵母 2-杂交检测, 同时发现在一个单一的屏幕上的许多瞬态和静态蛋白质相互作用的一个战略称为 DEEPN (动态富集评估蛋白质网络), 这采用高通量 DNA 测序和计算来跟踪编码相互作用伙伴的质粒种群的进化。在这里, 我们描述了定制的试剂和协议, 使 DEEPN 屏幕易于执行和经济高效。

Y2H 检测被广泛用于寻找蛋白质: 蛋白质相互作用和几个适应和系统已经开发。在大多数情况下, 同样的考虑, 帮助确保成功的这些以前的方法是重要的 DEEPN。一些重要的基准包括: 确保 DNA 结合领域融合蛋白的表达, 确保在 diploids 的低背景下, 他 + 生长在含有与空捕食质粒感兴趣的诱饵, 诱饵的交配效率高含有马来酵母与库含 MATalpha 酵母, 最后, 低严格的条件, 使人口增长的条...

Watch this Scientific Journal Video about A Yeast 2-Hybrid Screen in Batch to Compare Protein Interactions at JoVE.com

A Yeast 2-Hybrid Screen in Batch to Compare Protein Interactions

酵母 2-混合筛在批次中比较蛋白质相互作用

酵母 2-混合筛的分批处理可以直接比较多饵蛋白与高度复杂的猎物融合蛋白的相互作用。在这里, 我们描述了精制方法, 新试剂, 以及如何实现这些屏幕的使用。

Screening for protein-protein interactions using the yeast 2-hybrid assay has long been an effective tool, but its use has largely been limited to the ...

a-yeast-2-hybrid-screen-in-batch-to-compare-protein-interactions

Research

JoVE Journal

Biochemistry

13.2K 次观看.  University of Iowa. 酵母 2-混合筛的分批处理可以直接比较多饵蛋白与高度复杂的猎物融合蛋白的相互作用。在这里, 我们描述了精制方法, 新试剂, 以及如何实现这些屏幕的使用。

视频: A Yeast 2-Hybrid Screen in Batch to Compare Protein Interactions

Fluorescence Anisotropy as a Tool to Study Protein-protein Interactions

Protein-protein interactions play an essential role in the function of a living organism. Once an interaction has been identified and validated it is necessary to characterize it at the structural and mechanistic level. Several biochemical and biophysical methods exist for such purpose. Among them, fluorescence anisotropy is a powerful technique particularly used when the fluorescence intensity of a fluorophore-labeled protein remains constant upon protein-protein interaction. In this technique, a fluorophore-labeled protein is excited with vertically polarized light of an appropriate wavelength that selectively excites a subset of the fluorophores according to their relative orientation with the incoming beam. The resulting emission also has a directionality whose relationship in the vertical and horizontal planes defines anisotropy (r) as follows: r=(IVV-IVH)/(IVV+2IVH), where IVV and IVH are the fluorescence intensities of the vertical and horizontal components, respectively. Fluorescence anisotropy is sensitive to the rotational diffusion of a fluorophore, namely the apparent molecular size of a fluorophore attached to a protein, which is altered upon protein-protein interaction. In the present text, the use of fluorescence anisotropy as a tool to study protein-protein interactions was exemplified to address the binding between the protein mutated in the Shwachman-Diamond Syndrome (SBDS) and the Elongation factor like-1 GTPase (EFL1). Conventionally, labeling of a protein with a fluorophore is carried out on the thiol groups (cysteine) or in the amino groups (the N-terminal amine or lysine) of the protein. However, SBDS possesses several cysteines and lysines that did not allow site directed labeling of it. As an alternative technique, the dye 4&#39;,5&#39;-bis(1,3,2 dithioarsolan-2-yl) fluorescein was used to specifically label a tetracysteine motif, Cys-Cys-Pro-Gly-Cys-Cys, genetically engineered in the C-terminus of the recombinant SBDS protein. Fitting of the experimental data provided quantitative and mechanistic information on the binding mode between these proteins.

Protein interactions are at the heart of a cell&#39;s function. Calorimetric and spectroscopic techniques are commonly used to characterize them. Here we describe fluorescence anisotropy as a tool to study the interaction between the protein mutated in the Shwachman-Diamond Syndrome (SBDS) and the Elongation factor-like 1 GTPase (EFL1).

荧光各向异性作为一种工具来研究蛋白质相互作用

Protein-protein interactions play an essential role in the function of a living organism. Once an interaction has been identified and validated it is ...

科研

生物化学

Using Scaffold Liposomes to Reconstitute Lipid-proximal Protein-protein Interactions In Vitro

Studies of integral membrane proteins in vitro are frequently complicated by the presence of a hydrophobic transmembrane domain. Further complicating these studies, reincorporation of detergent-solubilized membrane proteins into liposomes is a stochastic process where protein topology is impossible to enforce. This paper offers an alternative method to these challenging techniques that utilizes a liposome-based scaffold. Protein solubility is enhanced by deletion of the transmembrane domain, and these amino acids are replaced with a tethering moiety, such as a His-tag. This tether interacts with an anchoring group (Ni2+ coordinated by nitrilotriacetic acid (NTA(Ni2+)) for His-tagged proteins), which enforces a uniform protein topology at the surface of the liposome. An example is presented wherein the interaction between Dynamin-related protein 1 (Drp1) with an integral membrane protein, Mitochondrial Fission Factor (Mff), was investigated using this scaffold liposome method. In this work, we have demonstrated the ability of Mff to efficiently recruit soluble Drp1 to the surface of liposomes, which stimulated its GTPase activity. Moreover, Drp1 was able to tubulate the Mff-decorated lipid template in the presence of specific lipids. This example demonstrates the effectiveness of scaffold liposomes using structural and functional assays and highlights the role of Mff in regulating Drp1 activity.

Using Scaffold Liposomes to Reconstitute Lipid-proximal Protein-protein Interactions In Vitro

This paper describes a method for assessing the interactions and assemblies of integral membrane proteins in vitro with various partner factors in a lipid-proximal environment.

使用脚手架脂质体重构脂质近端蛋白质 - 蛋白质相互作用<em&gt;体外</em

Studies of integral membrane proteins in vitro are frequently complicated by the presence of a hydrophobic transmembrane domain. Further complicating ...

Time-resolved ElectroSpray Ionization Hydrogen-deuterium Exchange Mass Spectrometry for Studying Protein Structure and Dynamics

Intrinsically disordered proteins (IDPs) have long been a challenge to structural biologists due to their lack of stable secondary structure elements. Hydrogen-Deuterium Exchange (HDX) measured at rapid time scales is uniquely suited to detect structures and hydrogen bonding networks that are briefly populated, allowing for the characterization of transient conformers in native ensembles. Coupling of HDX to mass spectrometry offers several key advantages, including high sensitivity, low sample consumption and no restriction on protein size. This technique has advanced greatly in the last several decades, including the ability to monitor HDX labeling times on the millisecond time scale. In addition, by incorporating the HDX workflow onto a microfluidic platform housing an acidic protease microreactor, we are able to localize dynamic properties at the peptide level. In this study, Time-Resolved ElectroSpray Ionization Mass Spectrometry (TRESI-MS) coupled to HDX was used to provide a detailed picture of residual structure in the tau protein, as well as the conformational shifts induced upon hyperphosphorylation.

Conformational flexibility plays a critical role in protein function. Herein, we describe the use of time-resolved electrospray ionization mass spectrometry coupled to hydrogen-deuterium exchange for probing the rapid structural changes that drive function in ordered and disordered proteins.

时间分辨电喷雾电离氢氘交换质谱用于研究蛋白质结构与动态

Intrinsically disordered proteins (IDPs) have long been a challenge to structural biologists due to their lack of stable secondary structure elements. ...

A Modified Yeast-one Hybrid System for Heteromeric Protein Complex-DNA Interaction Studies

Over the years, the yeast one-hybrid assay has proven to be an important technique for the identification and validation of physical interactions between proteins such as transcription factors (TFs) and their DNA target. The method presented here utilizes the underlying concept of the Y1H but is modified further to study and validate protein complexes binding to their target DNA. Hence, it is referred to as the modified yeast one-hybrid (Y1.5H) assay. This assay is cost effective and can be easily performed in a regular laboratory setting. Albeit using a heterologous system, the described method could be a valuable tool to test and validate the heteromeric protein complex binding to their DNA target(s) for functional genomics in any system of study, especially plant genomics.

The modified yeast one-hybrid assay described here is an extension of the classical yeast one-hybrid (Y1H) assay to study and validate the heteromeric protein complex-DNA interaction in a heterologous system for any functional genomics study.

用于异构蛋白复合物 -  DNA相互作用研究的修饰的酵母一杂交系统

Over the years, the yeast one-hybrid assay has proven to be an important technique for the identification and validation of physical interactions between ...

Biotinylated Cell-penetrating Peptides to Study Intracellular Protein-protein Interactions

Here we present a protocol to study intracellular protein-protein interactions that is based on the widely used biotin-avidin pull-down system. The modification presented includes the combination of this technique with cell-penetrating sequences. We propose to design cell-penetrating baits that can be incubated with living cells instead of cell lysates and therefore the interactions found will reflect those that occur within the intracellular context. Connexin43 (Cx43), a protein that forms gap junction channels and hemichannels is down-regulated in high-grade gliomas. The Cx43 region comprising amino acids 266-283 is responsible for the inhibition of the oncogenic activity of c-Src in glioma cells. Here we use TAT as the cell-penetrating sequence, biotin as the pull-down tag and the region of Cx43 comprised between amino acids 266-283 as the target to find intracellular interactions in the hard-to-transfect human glioma stem cells. One of the limitations of the proposed method is that the molecule used as bait could fail to fold properly and, consequently, the interactions found could not be associated with the effect. However, this method can be especially interesting for the interactions involved in signal transduction pathways because they are usually carried out by intrinsically disordered regions and, therefore, they do not require an ordered folding. In addition, one of the advantages of the proposed method is that the relevance of each residue on the interaction can be easily studied. This is a modular system; therefore, other cell-penetrating sequences, other tags, and other intracellular targets can be employed. Finally, the scope of this protocol is far beyond protein-protein interaction because this system can be applied to other bioactive cargoes such as RNA sequences, nanoparticles, viruses or any molecule that can be transduced with cell-penetrating sequences and fused to pull-down tags to study their intracellular mechanism of action.

This is a protocol to study intracellular protein-protein interactions based on the biotin-avidin pull-down system with the novelty of combining cell-penetrating sequences. The main advantage is that the target sequence is incubated with living cells instead of cell lysates and therefore the interactions will occur within the cellular context.

化细胞穿透肽研究胞内蛋白-蛋白质相互作用

Here we present a protocol to study intracellular protein-protein interactions that is based on the widely used biotin-avidin pull-down system. The ...

A Fluorescence Fluctuation Spectroscopy Assay of Protein-Protein Interactions at Cell-Cell Contacts

A variety of biological processes involves cell-cell interactions, typically mediated by proteins that interact at the interface between neighboring cells. Of interest, only few assays are capable of specifically probing such interactions directly in living cells. Here, we present an assay to measure the binding of proteins expressed at the surfaces of neighboring cells, at cell-cell contacts. This assay consists of two steps: mixing of cells expressing the proteins of interest fused to different fluorescent proteins, followed by fluorescence fluctuation spectroscopy measurements at cell-cell contacts using a confocal laser scanning microscope. We demonstrate the feasibility of this assay in a biologically relevant context by measuring the interactions of the amyloid precursor-like protein 1 (APLP1) across cell-cell junctions. We provide detailed protocols on the data acquisition using fluorescence-based techniques (scanning fluorescence cross-correlation spectroscopy, cross-correlation number and brightness analysis) and the required instrument calibrations. Further, we discuss critical steps in the data analysis and how to identify and correct external, spurious signal variations, such as those due to photobleaching or cell movement.
In general, the presented assay is applicable to any homo- or heterotypic protein-protein interaction at cell-cell contacts, between cells of the same or different types and can be implemented on a commercial confocal laser scanning microscope. An important requirement is the stability of the system, which needs to be sufficient to probe diffusive dynamics of the proteins of interest over several minutes.

This protocol describes a fluorescence fluctuation spectroscopy-based approach to investigate interactions among proteins mediating cell-cell interactions, i.e. proteins localized in cell junctions, directly in living cells. We provide detailed guidelines on instrument calibration, data acquisition and analysis, including corrections to possible artefact sources.

细胞细胞接触物蛋白质-蛋白质相互作用的荧光波动光谱测定

A variety of biological processes involves cell-cell interactions, typically mediated by proteins that interact at the interface between neighboring ...

A Fluorogenic Peptide Cleavage Assay to Screen for Proteolytic Activity: Applications for coronavirus spike protein activation

Enveloped viruses such as coronaviruses or influenza virus require proteolytic cleavage of their fusion protein to be able to infect the host cell. Often viruses exhibit cell and tissue tropism and are adapted to specific cell or tissue proteases. Moreover, these viruses can introduce mutations or insertions into their genome during replication that may affect the cleavage, and thus can contribute to adaptations to a new host. Here, we present a fluorogenic peptide cleavage assay that allows a rapid screening of peptides mimicking the cleavage site of viral fusion proteins. The technique is very flexible and can be used to investigate the proteolytic activity of a single protease on many different substrates, and in addition, it also allows exploration of the activity of multiple proteases on one or more peptide substrates. In this study, we used peptides mimicking the cleavage site motifs of the coronavirus spike protein. We tested human and camel derived Middle East Respiratory Syndrome coronaviruses (MERS-CoV) to demonstrate that single and double substitutions in the cleavage site can alter the activity of furin and dramatically change cleavage efficiency. We also used this method in combination with bioinformatics to test furin cleavage activity of feline coronavirus spike proteins from different serotypes and strains. This peptide-based method is less labor- and time intensive than conventional methods used for the analysis of proteolytic activity for viruses, and results can be obtained within a single day.

We present a fluorogenic peptide cleavage assay that allows a rapid screening of the proteolytic activity of proteases on peptides representing the cleavage site of viral fusion peptides. This method can also be used on any other amino acid motif within a protein sequence to test for the protease activity.

一种用于蛋白质溶解活性的荧光肽裂解检测: 冠状病毒穗蛋白活化的应用

Enveloped viruses such as coronaviruses or influenza virus require proteolytic cleavage of their fusion protein to be able to infect the host cell. Often ...

Analysis of Protein Folding, Transport, and Degradation in Living Cells by Radioactive Pulse Chase

Radioactive pulse-chase labeling is a powerful tool for studying the conformational maturation, the transport to their functional cellular location, and the degradation of target proteins in live cells. By using short (pulse) radiolabeling times (&lt;30 min) and tightly controlled chase times, it is possible to label only a small fraction of the total protein pool and follow its folding. When combined with nonreducing/reducing SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoprecipitation with (conformation-specific) antibodies, folding processes can be examined in great detail. This system has been used to analyze the folding of proteins with a huge variation in properties such as soluble proteins, single and multi-pass transmembrane proteins, heavily N- and O-glycosylated proteins, and proteins with and without extensive disulfide bonding. Pulse-chase methods are the basis of kinetic studies into a range of additional features, including co- and posttranslational modifications, oligomerization, and polymerization, essentially allowing the analysis of a protein from birth to death. Pulse-chase studies on protein folding are complementary with other biochemical and biophysical methods for studying proteins in vitro by providing increased temporal resolution and physiological information. The methods as described within this paper are adapted easily to study the folding of almost any protein that can be expressed in mammalian or insect-cell systems.

Here we describe a protocol for a general pulse-chase method that allows the kinetic analysis of folding, transport, and degradation of proteins to be followed in live cells.

放射性脉冲追逐对活细胞蛋白质折叠、迁移和降解的分析

Radioactive pulse-chase labeling is a powerful tool for studying the conformational maturation, the transport to their functional cellular location, and ...

Application of Biolayer Interferometry (BLI) for Studying Protein-Protein Interactions in Transcription

A transcription factor&#160;(TF) is a protein that regulates gene expression by interacting with the RNA polymerase, another TF, and/or template DNA. GrgA is a novel transcription activator found specifically in the obligate intracellular bacterial pathogen Chlamydia. Protein pulldown assays using affinity beads have revealed that GrgA binds two &#963; factors, namely &#963;66 and &#963;28, which recognize different sets of promoters for genes whose products are differentially required at developmental stages. We have used BLI to confirm and further characterize the interactions. BLI demonstrates several advantages over pulldown: 1) It reveals real-time association and dissociation between binding partners, 2) It generates quantitative kinetic parameters, and 3) It can detect bindings that pulldown assays often fail to detect. These characteristics have enabled us to deduce the physiological roles of GrgA in gene expression regulation in Chlamydia, and possible detailed interaction mechanisms. We envision that this relatively affordable technology can be extremely useful for studying transcription and other biological processes.

Application of Biolayer Interferometry BLI for Studying Protein-Protein Interactions in Transcription

Interactions of transcription factors (TFs) with the RNA polymerase are usually studied using pulldown assays. We apply a Biolayer Interferometry (BLI) technology to characterize the interaction of GrgA with the chlamydial RNA polymerase. Compared to pulldown assays, BLI detects real-time association and dissociation, offers higher sensitivity, and is highly quantitative.

生物层干涉测量(BLI)在转录中研究蛋白质-蛋白质相互作用的应用

A transcription factor&#160;(TF) is a protein that regulates gene expression by interacting with the RNA polymerase, another TF, and/or template DNA. GrgA ...

Use of Microscale Thermophoresis to Measure Protein-Lipid Interactions

The ability to determine the binding affinity of lipids to proteins is an essential part of understanding protein-lipid interactions in membrane trafficking, signal transduction and cytoskeletal remodeling. Classic tools for measuring such interactions include surface plasmon resonance (SPR) and isothermal titration calorimetry (ITC). While powerful tools, these approaches have setbacks. ITC requires large amounts of purified protein as well as lipids, which can be costly and difficult to produce. Furthermore, ITC as well as SPR are very time consuming, which could add significantly to the cost of performing these experiments. One way to bypass these restrictions is to use the relatively new technique of microscale thermophoresis (MST). MST is fast and cost effective using small amounts of sample to obtain a saturation curve for a given binding event. There currently are two types of MST systems available. One type of MST requires labeling with a fluorophore in the blue or red spectrum. The second system relies on the intrinsic fluorescence of aromatic amino acids in the UV range. Both systems detect the movement of molecules in response to localized induction of heat from an infrared laser. Each approach has its advantages and disadvantages. Label-free MST can use untagged native proteins; however, many analytes, including pharmaceuticals, fluoresce in the UV range, which can interfere with determination of accurate KD values. In comparison, labeled MST allows for a greater diversity of measurable pairwise interactions utilizing fluorescently labeled probes attached to ligands with measurable absorbances in the visible range as opposed to UV, limiting the potential for interfering signals from analytes.

Microscale thermophoresis obtains binding constants quickly at low material cost. Either labeled or label free microscale thermophoresis is commercially available; however, label free thermophoresis is not capable of the diversity of interaction measurements that can be performed using fluorescent labels. We provide a protocol for labeled thermophoresis measurements.